Dmitry E. Agafonov

Semiquantitative Proteomic Analysis of the Human Spliceosome via a Novel Two-Dimensional Gel Electrophoresis Method

ABSTRACT More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinity-purified human B, Bact, and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, Bact, and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosomes compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.

Molecular Architecture of the Human Prp19/CDC5L Complex

ABSTRACT Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosomes catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Protein-protein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of ∼20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.

Molecular architecture of the human U4/U6.U5 tri-snRNP

A human spliceosomal subcomplex The spliceosome is an RNA and protein molecular machine that cuts out introns from messenger RNAs. Agafonov et al. used cryo-electron microscopy to determine the structure of the largest intermediate subcomplex on the assembly pathway for the human spliceosome (see the Perspective by Cate). The structure shows substantial differences from the equivalent yeast complex. It also reveals how the subcomplex must dock onto the rest of the spliceosome and hints at the structural changes the complex must go through to form the mature spliceosome. Science, this issue p. 1416; see also p. 1390 The structure of the largest human spliceosome subcomplex reveals substantial differences from the equivalent yeast complex. [Also see Perspective by Cate] The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo–electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase–like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome’s catalytic RNA network.

Cryo-EM structure of a human spliceosome activated for step 2 of splicing.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2–U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.

HIS-24 Linker Histone and SIR-2.1 Deacetylase Induce H3K27me3 in the Caenorhabditis elegans Germ Line

ABSTRACT HIS-24 linker histone and SIR-2.1 deacetylase are involved in chromatin silencing in Caenorhabditis elegans. Depletion of SIR-2.1 results in cytoplasmic retention of HIS-24 in oocytes. However, the molecular working mechanisms of HIS-24 and SIR-2.1 are unclear. We show here a synergistic function of SIR-2.1 and HIS-24 that are together essential for maintenance of the H3K27me3 mark in the germ line of C. elegans. We demonstrate the synthetic effects of the two factors on brood size, embryogenesis, and fertility. SIR-2.1 and HIS-24 associate with the subtelomeric regions but apparently do not interact directly. We report that SIR-2.1 deacetylates H3K9 at subtelomeric regions and suggest that deacetylation of H3K9 is a prerequisite for H3K27 methylation. In turn, we found that HIS-24 specifically interacts with the histone H3 K27 region, when unmodified or in the trimethylated state. Overall, our data indicate that SIR-2.1 and HIS-24 contribute to the propagation of a specialized chromatin state at the subtelomeric regions and elsewhere in the genome.

Dynamic contacts of U2, RES, Cwc25, Prp8 and Prp45 proteins with the Pre-mRNA branch-site and 3' splice site during catalytic activation and step 1 catalysis in yeast spliceosomes.

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast Bact spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the Bact crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosomes protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity

The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.

The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction

The DEAH-box NTPase Prp43 and its cofactors Ntr1 and Ntr2 form the NTR complex and are required for disassembling intron-lariat spliceosomes (ILS) and defective earlier spliceosomes. However, the Prp43 binding site in the spliceosome and its target(s) are unknown. We show that Prp43 fused to Ntr1s G-patch motif (Prp43_Ntr1GP) is as efficient as the NTR in ILS disassembly, yielding identical dissociation products and recognizing its natural ILS target even in the absence of Ntr1’s C-terminal-domain (CTD) and Ntr2. Unlike the NTR, Prp43_Ntr1GP disassembles earlier spliceosomal complexes (A, B, Bact), indicating that Ntr2/Ntr1-CTD prevents NTR from disrupting properly assembled spliceosomes other than the ILS. The U2 snRNP-intron interaction is disrupted in all complexes by Prp43_Ntr1GP, and in the spliceosome contacts U2 proteins and the pre-mRNA, indicating that the U2 snRNP-intron interaction is Prp43’s major target. DOI: http://dx.doi.org/10.7554/eLife.15564.001

Stable tri-snRNP integration is accompanied by a major structural rearrangement of the spliceosome that is dependent on Prp8 interaction with the 5‘ splice site.

Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.

Identification of a small molecule inhibitor that stalls splicing at an early step of spliceosome activation

Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation. DOI: http://dx.doi.org/10.7554/eLife.23533.001